Preservation of naive-phenotype CD4+ T cells after vaccination contributes to durable immunity

Memory T cells are conventionally associated with durable recall responses. In our longitudinal analyses of CD4+ T cell responses to the yellow fever virus (YFV) vaccine by peptide-MHC tetramers, we unexpectedly found CD45RO–CCR7+ virus-specific CD4+ T cells that expanded shortly after vaccination and persisted months to years after immunization. Further phenotypic analyses revealed the presence of stem cell–like memory T cells within this subset. In addition, after vaccination T cells lacking known memory markers and functionally resembling genuine naive T cells were identified, referred to herein as marker-negative T (TMN) cells. Single-cell TCR sequencing detected expanded clonotypes within the TMN subset and identified TMN TCRs shared with memory and effector T cells. Longitudinal tracking of YFV-specific responses over subsequent years revealed superior stability of TMN cells, which correlated with the longevity of the overall tetramer+ population. These findings uncover additional complexity within the post-immune T cell compartment and implicate TMN cells in durable immune responses.


Introduction
Functional immunological memory underlies the protective efficacy of vaccines against subsequent infections (1,2).However, the reason why protection from some vaccines lasts decades while others wane after a few months remains unknown.A crucial aspect of immune memory involves CD4 + T cells (3).CD4 + T cells provide key signals for B cell maturation and high-affinity antibody production (4).They are also needed to support the expansion and maintenance of functional CD8 + T cells and can directly contribute to anti-viral effects (4)(5)(6).Past studies in mice and humans have identified naïve-like antigen-experienced T cells with superior longevity and plasticity as a source of durable memory (7)(8)(9).Broadly categorized as stem cell-like memory T cells (TSCM), these cells phenotypically resemble naïve T cells by positive CCR7 and CD45RA or negative CD45RO expression, yet they display differentiation markers such as CD95, CXCR3, and CD49d (9)(10)(11).In people immunized with the highly efficacious and durable Yellow Fever Virus (YFV) vaccine, class I tetramer analyses identified TSCM as the predominant phenotype of virus-specific CD8 + T cells greater than 8 years after vaccination (10,12).
The durability of CD4 + T cell memory is less understood.Although capable of differentiating into TSCM cells (13)(14)(15)(16), CD4 + T cells are generally less responsive to homeostatic cytokines IL-7 and IL-15 (17)(18)(19), which augment TSCM differentiation in cultured CD8 + T cells (20).Here, we examined virus-specific CD4 + T cells after YFV vaccination to delineate key features of durable CD4 + T cell responses.YFV-specific CD4 + T cells were identified and tracked longitudinally by direct ex vivo class II peptide-MHC (pMHC) tetramers staining.We showed the presence of various memory subsets within YFV-specific CD4 + T cells several months after vaccination, including TSCM cells.Unexpectedly, about a quarter of CD45RO -CCR7 + tetramer-labeled T cells, lacked CD95, CXCR3, CD11a, and CD49d expression, distinguishing them from TSCM cells.These marker negative T cells (TMN) were a part of expanded clonotypes and shared TCR sequences with memory and effector T cells, suggesting in vivo antigen responses.TMN cells showed minimal decay over years and correlated with the stability of tetramer + populations.Our findings expand the current definition of antigenexperienced T cells to include those that retained an undifferentiated phenotype.These TMN cells may contribute to durable immunity.

Detection of naïve-like CD4 + T cells after YFV vaccination
We had previously performed a longitudinal study of YFV-specific CD4 + T cells to evaluate the impact of preexisting repertoire on T cell responses to primary immunization with the YFV vaccine (21).Starting with this dataset, we examined features of memory T cells that developed at least 7 months after vaccination.This showed that approximately half of the YFV-specific memory pool consisted of central memory T cells (TCM), with about 21% of tetramer + cells retaining a naïve-like CD45RO -CCR7 + phenotype (Fig. 1A-B).Proportionally, the abundance of CD45RO -CCR7 + subset was highest before vaccination, decreased initially post-vaccination, then reaccumulated several months later (Fig. 1C).Quantified as cells per million CD4 + T cells, CD45RO - CCR7 + tetramer + cells quickly increased and reached a peak approximately 1 month after vaccination (Fig. 1D).The frequency of CD45RO -CCR7 + T cell subset did not differ significantly by donor age and was associated with the robustness of the response (Fig. S1A-C).At the memory time point, CD45RO -CCR7 + YFVspecific T cells were more abundant in tetramer + populations that reached a higher frequency and correlated with efficient recruitment of memory cells (Fig. 1E-F).These data suggest that CD45RO -CCR7 + CD4 + T cells is a feature of an effective T cell response.

Post-immune T cells are heterogeneous and include a differentiation marker negative subset
We hypothesized that the post-vaccine CD45RO -CCR7 + subset largely consisted of TSCM cells as in CD8 + T cells (10,12).To test this, we performed tetramer staining on 28 YFV-specific CD4 + populations from 7 individuals, recognizing 16 unique epitopes with antibodies against TSCM-associated markers, CXCR3, CD95, CD11a, and CD49d (Tables S1 and S2).Staining with this broader antibody panel on blood collected 7 to 48 months after vaccination indeed identified CD45RO -CCR7 + tetramer + T cells that expressed one or more TSCM markers.However, we noted that a portion of CD45RO -CCR7 + CD4 + T cells remained negative for CXCR3, CD95, CD11a, and CD49d expression (Fig. 2A, S2A-D).To gain further insights into the heterogeneity within the CD45RO-CCR7 + subset, we combined 1465 YFV-specific CD4 + T cells from one donor and visualized combinatorial antibody staining on UMAP using the Spectre pipeline (22).This identified regions with low CD45RO and high CCR7 signals, which encompassed a CXCR3 + (cluster 0) and a TSCM marker negative population (cluster 4) (Fig. 2B-D).We defined CD45RO -CCR7 + cells lacking any measured differentiation markers as marker-negative T cells (TMN) and classified those expressing at least one of CXCR3, CD95, CD11a, or CD49d as TSCM cells (Table S3).We then performed manual gating and used Boolean combinations of these gated cells to subdivide CD45RO -CCR7 + subset into TMN and various TSCM combinations (Fig. S2B-D).On average, 27% of the CD45RO -CCR7 + subset consisted of TMN cells (Fig. 2E).Among TSCM cells, approximately a quarter expressed only one differentiation marker, with CXCR3 being the most common (Fig. 2E-F).Finding antigen-specific T cells that do not express known memory or TSCM markers after a clear prior exposure was unexpected.To test if TMN cells functionally behave like antigen-experienced T cells despite lacking surface markers of differentiation, we treated post-vaccine PBMCs with PMA and ionomycin for 4 to 5 hours.Antigen-specific T cells were captured by tetramers, divided into distinct phenotypic subsets, and analyzed for TNF-a and IFN-g production.This showed that post-immune TMN subset produced significantly less cytokines compared to memory T cells within the same tetramer + population (Fig. 2G-H).Thus, YFV vaccination induced a diverse post-immune repertoire that included CD4 + TSCM cells and a naïve-like TMN population that lacked phenotypic and functional features of antigen experience.

Virus-specific TMN cells respond to antigens
We were intrigued by the existence of virus-specific T cells that retained a naïve functional phenotype after vaccination.Past studies have identified non-stimulatory TCR interactions that decoupled T cell activation from ligand binding (23).The impaired ability to respond productively to antigens may be one reason why some tetramer-labeled T cells retained a naïve phenotype.To investigate this possibility, we quantified TMN, TSCM, and TCM cells for differences in their functional avidity by peptide stimulation.YFV-specific T cell clones were generated using samples from two donors obtained 7 to 8 months after YFV vaccination.Among the 48 clones that grew, 40 clones (90%) had the correct specificity by tetramer re-staining and/or response to peptides (Fig. 3A-B, S3A).We did not identify peptide-nonresponsive T cells as all clones that were stained with tetramers responded to peptide stimulation.To determine if TMN cells might be harder to activate due to a lower functional avidity, we divided the clones according to their direct ex vivo phenotype and selected 5 clones each from TCM, TSCM, TMN groups for further analyses.YFV-specific clones were stimulated with decreasing concentrations of the cognate peptide and analyzed for response by cytokine production (Fig. S3B).TMN-derived clones responded similarly to peptides by TNF-a production, with no significant differences in maximal effective peptide concentration (EC50) values between groups (Fig. 3C-D).T cell clones, regardless of their ex vivo phenotypes, also produced similar levels of IFN-g, IL-2, and had comparable TNF-a + IFN-g + IL-2 + co-expression (Fig. 3E).In addition, we evaluated the proliferative capacity of TMN, TSCM, and TCM-derived clones by CellTrace Violet (CTV) dilution and observed no significant differences in the proliferative response to peptide stimulation (Fig. 3F-G, Fig. S3C).Thus, TCR-ligand engagement is likely intact for vaccine-specific T cells that retained a naïve phenotype after vaccination.

Antigen-experienced TMN cells
While TMN cells respond well to antigens in vitro, it remains possible for them to be less competitive in resource-limiting environments.To investigate this, we reason that we can use TCR sequences to infer stimulation and proliferative response in vivo.Because T cell progenies originating from a T cell express identical TCR sequences, we can further leverage these sequences as molecular barcodes to investigate the clonal relationship between distinct phenotypic subsets.However, capturing sufficient numbers of TMN cells was challenging due to their limited number within the available blood samples.To overcome this problem, we generated new tetramers using affinity-matured HLA-DR monomers containing mutations that enhanced CD4 binding to improve the overall capture efficiency (24).When compared to the wild-type (wt) DR, these tetramers stained a larger population of T cells without significantly skewing the phenotypic proportions (Fig. S4A-C).In total, we sorted single cells from 5 tetramer-labeled populations and obtained TCR sequences from 607 YFV-specific CD4 + T cells after amplification and sequencing (Fig. 4A, Table S4).Consistent with clonal expansion after vaccination, over 70% of the sequences were identified in multiple tetramer-sorted T cells.Among expanded sequences, 25 to 52% were abundant and found in at least 10 individual T cells (Fig. 4B).Most T cells displayed a TCM or TEM phenotype based on antibody staining at the time of sorting.TMN phenotype was infrequent, expressed by 3 to 4% of sequenced T cells and confined to the two most extensively sequenced populations recognizing YF45.Consistent with in vivo expansion, TMN cells did not preferentially express unique TCRs, but rather, they were distributed across various clone sizes (Fig. 4C).We focused the subsequent analyses on YF45-specific T cells that included the TMN subset.Early post-vaccine measurements of YF45-specific T cells from HD2 and HD3 showed that both populations had generated robust responses to the YFV vaccine (Fig. 4D) (21).In agreement with an antigen-driven response, TMN cells contained expanded clonotypes and shared overlapping sequences with various memory subsets (Fig. 4E-F,  S4E).In separately generated T cell clones from the same individuals, TMN-derived clones expressed TCRs that matched the sequences from ex vivo sorted T cells of diverse clone sizes and phenotypes (Fig. S4F).
The presence of shared TCR sequences with memory T cells, together with clonal expansion, suggest that TMN cells had encountered and responded to antigens.Alternatively, clonotype sharing between memory and naïve cells may be explained by the presence of multiple naïve T cells with the same TCR, where a portion did not encounter YFV antigen and remained naïve.To investigate this possibility, we examined the pre-vaccination repertoire of YF45-specific T cells in these individuals to determine if pre-exposure T cells expressing TMNassociated TCRs were abundant before vaccination (21).The changes in clonal dynamics were assessed by tetramer staining, sorting, and sequencing the TCRs of YF45-specific T cells from blood collected 14 days after vaccination.Of note, the sampling depth at day 14 was much shallower due to limited sample availability.Ten to twenty million PBMCs was used for tetramer staining versus over a hundred million CD4 + T cells were used to capture rare precursor T cells in the pre-vaccine sample.In total, we examined TCR sequences from 129 precursor T cells and 238 effector T cells (Fig. 5A, Table S5).Before vaccination, no pre-vaccine TCRs matched TMN-derived TCRs from HD2 and only one sequence was identified in HD3.This shared TCR mapped to a unique sequence and not to the expanded pre-existing clonotypes in this individual (Fig. 5B-C).Fourteen days following vaccination, matched TCR frequencies increased to 7% in HD2 and 23% in HD3, capturing a total of 7 clonotypes (Fig. 5B-C).The majority of matched clonotypes were expanded and expressed by a variety of differentiated cells (Fig. 5D).Among T cells without a TMN match, two cells in HD3 were TMN-like and lacked differentiation marker expression.One of these cells was found as a part of an expanded clone at day 14 and shared the same TCR with multiple memory T cells in the day 210 sample (Fig. 5E, Table S5).Additionally, the post-vaccine day 14 blood from this donor contained three CD45RO -CCR7 + T cells without additional differentiation marker staining (Table S5).
Human T cells stimulated with cytokines can maintain a naïve phenotype (25).Our data suggest that this can also occur with antigen-specific responses.To broaden the analyses, we compared the frequency of CD45RO -CCR7 + T cells in various YFV-specific populations before and after YFV vaccination (Fig. 5F).This showed an increase in CD45RO -CCR7 + tetramer + cells post-exposure, which included diverse ratios of TSCM and TMN cells (Fig. 5G).We focused on the TMN subset as they resembled unstimulated naïve T cells.If there is no mechanism to replenish the naïve repertoire and/or hold on to a naïve phenotype, we expected a decrease in naïve cell frequency following an immune response as responding T cells acquire differentiated states.However, contrary to this, the averaged post-vaccine TMN cell frequency was comparable to the frequency of naive cells before vaccination (Fig. 5H).To examine population-level differences, we divided post-vaccine TMN frequency of each population by its initial naïve T cells frequency before vaccination.This revealed lower TMN frequencies in two-third of the populations, while the remaining third showed an increase (Fig. 5I).The absence of a reduction in naïve T cells did not indicate a lack of responsiveness to the vaccine.Instead, among the ten populations that gained naive T cells, there was a higher averaged fold-change in total tetramer + frequency before and after vaccination (Fig. 5J).Collectively, these data suggest TMN subset comprises T cells that had responded to vaccination.

TMN cells contribute to durable memory
While memory T cells are essential for generating rapid recall responses, naïve T cells are known for their long life-span and regenerative potential (26,27).Therefore, we hypothesized that TMN cells promote the durability of antigen-specific responses after vaccination.To test this idea, we analyzed additional time points from five donors with longitudinal PBMCs collected up to 6.7 years after YFV vaccination (Fig. 6A, Fig. S5A).Past modeling of cellular turnover suggests that different phenotypic subpopulations undergo separate and distinct in vivo dynamics (28,29).To evaluate the stability of individual phenotypic subsets, we subdivided 19 YFVspecific populations according to TMN, TSCM, TCM, TEM, and TEMRA phenotypes based on CD45RO, CCR7, CD95, CXCR3, CD11a, and CD49d expression.Their time-dependent change was quantified as a fitted slope using a mixed-effects exponential decay model.This revealed different rates of decay between cells in distinct differentiation states.CD4 + TEM cells had the largest negative slope, indicating the greatest decrease over time.In contrast, TMN cells exhibited remarkable stability, with no discernible decline observed during the follow-up period.The stability of the TMN subset significantly surpassed that of other phenotypic subsets, including TSCM and TCM cells, which are typically considered to be long-lived (Fig. 6B).
Next, we examined the decay kinetics of the overall tetramer + populations.Because some data were generated before switching to modified DR, paired analyses by wildtype and modified tetramers on the same blood sample were used to generate an equation for normalizing the frequencies across experiments (Fig. S4D).Among the five donors followed longitudinally, two received one YFV dose as typical for the YFV vaccine, while three received a second YFV vaccine 7 months to a year after the initial dose (Table S6).Revaccination was originally a part of an IRB-approved protocol to study recall response.While this strategy did not effectively induce acute T cell responses, likely due to YFV neutralization by antibodies generated from the first vaccination, we investigated whether it might still impact long-term T cell dynamics.We divided tetramer + populations based on donors' vaccine doses.Consistent with the longevity of YFV vaccine-mediated protection, YFV-specific CD4 + T cells displayed an average half-life (t1/2) of close to 4 years after one YFV immunization (Fig. 6C).This durable response was further stabilized after re-exposure in the two-dose group (Fig. S5B).TMN cell frequencies did not significantly differ between the one-dose and two-dose groups (Fig. S5C).Given the wide variation in TMN cells, we divided tetramer + populations based on TMN frequency into top and bottom halves (Fig. 6D).This showed that populations with more TMN cells were more stable compared to populations in the bottom TMN group (Fig. 6E).The TMN frequency within a given virus-specific population further positively correlated with the stability of the overall population (Fig. 6F).By contrast, we did not find significant differences between high and low groups based on TSCM, TCM, TEM, and TEMRA frequencies (Fig. S5D).On the phenotypic level, all tetramer + populations contained various memory subsets, but the top TMN group was more phenotypically diverse.We divided populations based on the first TMN frequency obtained within the 1-2 years after YFV vaccination and showed that populations with more TMN cells exhibited greater diversity of differentiation states over time as measured by the Shannon diversity index (Fig. 6G-H).Collectively, these data highlight the stability of the TMN subset and uncover their association with durable and diverse T cell memory after YFV vaccination.

Discussion
A T cell is typically referred to as naïve if it has not yet encountered its specific cognate antigen(s).However, a clear antigenic history is often not available in human studies, and thus specific surface markers are commonly used to infer antigen experience (11,30).Here, we examined CD4 + T cell memory to YFV vaccination to define key features of durable memory by direct ex vivo class II tetramer staining and enrichment.This showed a diverse memory pool comprised of various differentiation states after YFV vaccination.Surprisingly, we also uncovered an increase in naïve phenotype YFV-specific T cells after vaccination.
Deeper phenotypic analyses showed that a portion of CD45RO -CCR7 + cells was previously described TSCM cells.The remaining cells did not express known surface markers of T cell differentiation that were tested.We evaluated several potential explanations for their origin.Our initial idea that TMN cells were unresponsive was incorrect.Peptide-stimulated TMN-derived T cell clones produced robust levels of cytokines at comparable EC50 as T cell clones generated from memory cells.The presence of expanded TCR clones within the TMN subset further suggest that some TMN cells had seen and responded to antigens in vivo.The observation that TMN cells, memory, and effector T cells have overlapping TCR repertoires suggests shared precursors among these subsets and provide additional support to the idea that some TMN cells are antigen-experienced as their common precursors divided at least once in response to antigen.This extent of overlap would not be expected if clonal expansion within TMN subset were driven by different processes, such as cytokine-mediated and/or bystander response.Our interpretation that some expanded antigen-experienced cells exist within the TMN population is further supported by increased naïve T cell frequency following immunization.We show that, despite recruitment into effector responses, the frequency of naïve T cell frequency recognizing YFV is largely unchanged.Among populations analyzed pre-and post-immunization, approximately a third gained more naïve phenotype T cells in the form of TMN cells above their pre-vaccine baseline.Probabilistically, it is also unlikely to capture TMN cells 14 days after vaccination if they had not expanded, since the same clonotype was not detected before vaccination despite analyzing many more cells.
In addition to an antigen-experienced subset, some TMN cells could be genuine naïve T cells.As an alternative possibility, there might be multiple naïve cells of the same clonotype but only a subset responded to the vaccine.Incomplete response may have occurred because some YFV-specific T cells were not at the right place or time to respond to the immunogen.Other cells may not have received enough of the appropriate signal to undergo expansion and/or differentiation (33).This model could be consistent with our TCR data but does not explain why certain specificities contain higher frequencies of naïve T cells after vaccination.An intriguing alternative for this increase might involve ongoing thymic activities.Although thymic output for new T cells declines with age, recent thymic emigrants remain detectable in adults (34)(35)(36).Newly produced T cells might add to the peripheral TMN pool to increase the abundance of YFV-specific naïve T cells after vaccination.TMN cells from various sources could contribute to the naïve repertoire after antigen exposures, varying in their impact depending on antigen-specificity, the type of stimulation, age and other individual characteristics.
Only a few select vaccines are capable of mediating life-long protection.How durable immunological memory is maintained remains a key unresolved question.While Memory T cells are the cornerstone of protective immunity by virtue of their ability to rapidly initiate a functional response to pathogen rechallenge, naïve T cells possess superior self-renewal capacity and differentiation plasticity (3,26,37).Irrespective of how TMN cells might have originated, we asked whether TMN cells are related to the longevity of T cell response.Our findings revealed remarkable stability of the TMN population, showing minimal decay for nearly 7 years.These stable T cells could potentially support the longevity of the overall immune response, extending it beyond the lifespan of individual memory T cells.Consistent with this model, TMN cells are more abundant in durable CD4 + populations that are stable over time.Based on the diverse memory phenotypes in TMN-enriched populations, we further speculate that TMN cells have the potential to differentiate into multiple states, thereby contributing to the phenotypic diversity of T cell memory.
In summary, our findings highlight the complexity within the post-immune T cell compartment and add to our understanding of the diverse spectrum of T cells exhibiting naïve features (27,38).Using an antigen-specific approach, our data suggest that some cells considered naïve by phenotypic criteria are actually antigenexperienced.As a whole, post-immune TMN cells are remarkably stable.Understanding how TMN cells are generated, maintained, and work alongside memory cells in providing long-lasting immune protection could aid the future development of improved vaccine strategies.

Limitation of study
We did not further subset TMN cells to evaluate potential heterogeneity in gene-expression, epigenetic landscape, or functional attributes due to the limitations on cell numbers.Future studies using models allowing manipulation of TMN cells will be needed to build on their association with durable immunity and establish causation.As our analyses are focused on CD4 + T cell responses to YFV in a few healthy individuals, larger studies involving additional vaccines will be important to strengthen our findings and to understand how the TMN subset influences vaccine durability across different age and disease settings.

Acknowledgments:
We thank our study subjects for their participation.

Competing interests:
The authors declare no competing interests.

Sex as a biological variable
Both male and female participants were included in this study.Study volunteers were recruited in the order of participation without restrictions based on the sex.

Human samples
This study uses cryopreserved cells stored in fetal bovine serum (FBS) with 10% DMSO from an ongoing vaccine study at the University of Pennsylvania (21).This study includes 7 healthy adult participants with no prior YFV exposure who received one or two doses of the 17D live-attenuated YFV vaccine (YF-VAX®, Sanofi Pasteur).Individuals older than 65 were excluded.As YFV vaccine provides life-long protection against YFV, re-vaccination with the attenuated 17D-204 strain is not expected to increase risk.Five participants were followed longitudinally for 2 to 6.7 years after vaccination.All samples were de-identified and obtained with IRB regulatory approval from the University of Pennsylvania.Subject characteristics are shown in Table S1.

Protein expression and tetramer production
HIS-tagged HLA-DRA/B1*0301, 0401, 0407, and 1501 protein monomers of wild type (wt) sequence or with L112W, S118H, V143M, T157I mutations (24) were produced by Hi5 insect cells and extracted from culture supernatant using Ni-NTA (Qiagen).HLA-DR monomers were biotinylated overnight at 4 o C using BirA biotin ligase (Avidity) and purified by size exclusion chromatography using Superdex 200 size exclusion column (AKTA, GE Healthcare).Biotinylation was confirmed by gel-shift assay.Peptide exchange and tetramerization for wildtype and modified affinity-matured DR were performed using standard protocols as previously described (39,40).In brief, HLA-DR proteins were incubated with thrombin (Millipore) at room temperature for 3 -4 hours and exchanged with peptides of interest in 50-fold excess at 37 o C for 16 hours.Peptide-loaded HLA-DR monomers were incubated with fluorochrome-conjugated streptavidin at 4 -5: 1 ratio for 2 min at room temperature, followed by a 15 min incubation with an equal volume of biotin-agarose slurry (Millipore).Tetramers were buffered exchanged into PBS, concentrated using Amicon ULTRA 0.5ml 100KDa (Millipore), and kept at 4 o C for no more than 2 weeks prior to use.

Ex vivo T cell analyses and cell sorting
Phenotypic analyses and frequency quantification: Tetramer staining was performed on at least 10 million PBMCs with 5 ug of tetramers in 100 µl reaction for 1 hour at room temperature as previously described (21,40,41).Tetramer-tagged cells were enriched by adding anti-fluorochrome and anti-HIS MicroBeads (MiltenyiBiotec).The mixture was passed through LS columns (MiltenyiBiotec).Column-bound cells were washed and eluted according to manufacturer protocol.For antibody staining, the enriched samples were stained with viability dyes and exclusion markers (anti-CD19 and anti-CD11b, Table S7), along with combinations of surface markers as specified in the experiments (anti-CD3, anti-CD4, anti-CD45RO, anti-CCR7, anti-CD11a, anti-CD95, anti-CD49d, and anti-CXCR3, Table S7), in 50 to 100ul of FACS buffer (PBS plus 2% FCS, 2.5mM EDTA, 0.025% Sodium Azide) for 30 minutes at 4 o C. Samples were fixed with 2% paraformaldehyde and acquired by flow cytometry using LSRII (BD).Data analyses were performed by FlowJo (BD).The Boolean tools was used to define TSCM and TMN cells within the CD45RO -CCR7 + subset.Frequency calculation was obtained by mixing 1/10th of samples with 200,000 fluorescent beads (Spherotech) for normalization.
For longitudinal experiments involving both wt and modified DR, paired data from wt and modified DR, with a minimum of two data points per time point for each specificity, were used to derive the equation for normalization: log2(Freqmodified)=3.72+0.35* log2(Freqwt) (Fig. S4D).Frequencies generated by wt tetramers that were below the normalized values were adjusted.Mixed effects exponential decay models were used to analyze longitudinal changes in antigen-specific T cell populations and estimate the corresponding slopes.These models were implemented in MonolixSuite 2021R1 (Lixoft) and fitted to data after vaccination.Initial T cell specificity values were lognormally distributed, exponential decay rates were normally distributed, and lognormal multiplicative error was used.The estimation of the population parameters was performed using the Stochastic Approximation Expectation-Maximization (SAEM) algorithm.Half-lives were calculated as ln(2)/k, where the corresponding k values represented the estimated exponential decay rate constants.Estimated decay rates were converted into slopes as -k.
For multi-dimensional analyses, a total of 1465 manually gated tetramer + cells were exported from FlowJo, read into R by flowCore, and combined into one single dataset for subsequent data processing and analyses using the Spectre package in R (22).Staining intensities were converted using Arcsinh transformation with a cofactor of 200.Batch alignment was performed using the CytoNorm (42).Clustering was performed using Phenograph with nearest neighbors set to 55 (k = 55) (43).UMAP was used for dimensional reduction and visualization (44).
Function response: T cells were rested overnight, followed by 4 -5 hours of stimulation by phorbol myristate acetate (PMA, 5 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) in the presence of monensin (2 uM, Sigma) and Brefeldin A (5 ug/ml, Sigma).Tetramer and surface antibody staining were performed as above.Intracellular staining with antibodies to TNF-a, IFN-g, CD3 and CD4 (Table S7) was performed following BD Cytofix/Cytoperm Fixation/Permeabilization Kit according to manufacturer protocol (BD).
Cell sorting: Around 60 million CD3 + or CD4 + T cells were used and stained with up to 10 ug of each tetramer in a 100 ul reaction.Antibody staining was performed as above without fixation.Individual tetramer-labeled cells were isolated for TCR sequencing or T cell cloning by index sorting using the purity mode on FACS Aria (BD).
DCs generation: Monocytes from HLA-DR allele-matched donors were isolated using negative enrichment kits (RosetteSep Human Monocyte Enrichment Cocktail, StemCell).10 million cryopreserved monocytes were cultured in 15 ml DC media (RPMI 1640 plus Glutamine,10% FCS, 1X Pen/Strep, 10 mM HEPES) in the presence of 100 ng/ml GM-CSF and 500 U/ml IL-4.Three days later, half the culture media was replaced with fresh DC media with 100 ng/ml GM-CSF, 500 U/ml IL-4, and 0.05 mM 2-mercaptoethanol.Cells in suspension were harvested at 5 to 6 days and added to a flat-bottom 96-well plate at 25,000 DCs per well.DCs were treated with 100 ng LPS and peptides (0.00001 ug/ml to 10ug/ml) for 16 hours and replenished with fresh media before co-culturing with T cells.
Stimulation of T cell clones: T cell clones were rested overnight in fresh media without IL-2 and added to wells containing matured DCs at 1:1 ratio in the presence of monensin (2 uM, Sigma) and Brefeldin A (5 ug/ml, Sigma).After 5 hours, cells were transferred into a new 96-well round bottom plate, washed once with FACS buffer, and stained with viability dyes, exclusion markers (anti-CD19 and anti-CD11b) for 30 minutes at 4°C.Intracellular staining with antibodies to TNF-a, IFN-g, IL-2, CD3 and CD4 (Table S7) was performed following BD Cytofix/Cytoperm Fixation/Permeabilization Kit according to manufacturer protocol (BD).Half maximal effective concentration (EC50) was determined using the percentage of T cell clones that produced TNF-a in response to decreasing peptide concentrations (10, 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001 ug/ml).A nonlinear fit without constraint was applied to log-transformed concentration using the equation Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) in Prism (GraphPad).For proliferation assay, T cell clones were labeled with 1:1000 diluted CellTrace Violet (CTV, ThermoFisher Scientific) following manufacturer protocol.The CTV-stained cells were rested in fresh media without IL-2 for 16 hours.25,000 rested T cells were cocultured with DC pulsed with 10 ug/ml cognate peptides or treated with PHA as a positive control (1:100, ThermoFisher).After 5 days, cells were harvested and stained with viability dyes and surface antibodies (anti-CD19, anti-CD11b, anti-CD3, and anti-CD4 for 30 minutes at 4°C followed by fixation with 2% paraformaldehyde.Samples were acquired by flow cytometry using LSRII (BD) and analyzed by FlowJo (BD).

Single-cell TCR sequencing and analyses
Single-cell TCR Sequencing by nested PCRs was performed using the primer sets and the protocol as previously described (21,45).In brief, reverse transcription was performed with CellsDirect One-Step qRT-PCR kit according to the manufacturer's instructions (CellsDirect, Invitrogen) using a pool of 5' TRVB-region specific primers and 3' C-region primers.The cDNA library was amplified using a second set of multiple internally nested V-region and C-region primers with HotStarTaq DNA polymerase kit (Qiagen).The final PCR reaction was performed on an aliquot of the second reaction using a primer containing common base sequence and a third internally nested Cb primer.PCR products were gel purified (Qiagen) and sequenced on Novaseq 6000 platform (Illumina).TCR sequences were pre-processed as previously described (21).In brief, forward and reverse reads were converted into one paired end read using pandaseq (46).Data were demultiplexed by the unique combination of plate, row, and column barcodes.Consensus TCRβ sequences were identified using the V(D)J alignment software MiXCR (47).A threshold of a read count of 200 reads per sequence was applied to the consensus sequences.If more than one TCRa or TCRb chain passes this criterion we retain the dominant TCRb and the two TCRa chains with the highest read count.For data obtained from cells several months after vaccination, we additionally require phenotypic annotation based on antibody staining from index sort data.Data were excluded if phenotypic information was not retained or ambiguous.For downstream analyses, data wrangling was performed using the tidyverse package.TCRs were matched by TCRb if only the beta chain was available, or by TCRb plus at least one TCRa if alpha chain(s) were called.Circos plots were made using the circlize package of R software (48).

Statistics
Normality was assessed using D'Agostino-Pearson test.Spearman was used if either of the two variables being correlated was non-normal.Otherwise, Pearson was used to measure the degree of association.Least squares linear regression was used to calculate the best-fitting line.Statistical comparisons were performed using two-tailed Student's t-test, paired t-test, Welch's one-way ANOVA, repeated measures one-way ANOVA, two-way ANOVA, or mixed effects model.A p-values of <0.05 was used as the significance level and adjusted if multiple comparisons were performed.Statistical analyses were performed using GraphPad Prism.Lines and bars represent the mean and variability is represented by the standard error of the mean (SEM).* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Study approval
All participants have given written informed consent.The study was approved by the Institutional Review Board at the University of Pennsylvania, Philadelphia (approval 820884).The ratio is defined as a gain if above 1 and a loss if below 1. Pie chart shows the numbers of populations that had gained or lost naïve T cells after vaccination.(J) Plot shows the fold-change in tet + frequency, from the pre-vaccine baseline to the memory time point, for populations that had gained or lost naïve cells.For (F), (H), and (J), n = 27 tetramer + populations.Welch's t-test was performed.Welch's t-test was performed for (E) and (H).For (F), Spearman correlation was performed.

Figure 1 :
Figure 1: Identification of CD45RO -CCR7 + virus-specific CD4 + T cells after YFV vaccination.(A) Direct ex vivo tetramer and antibody staining of a representative YFV tetramer + (tet + ) population using blood collected about 7 months after YFV vaccination.(B) The percentage of YFV tet + T cells with the indicated combination of CD45RO and CCR7 expression.Plot summarizes data from 36 specificities 7 to 34 months after YFV vaccination from 7 donors.(C-D) The abundance of CD45RO -CCR7 + YFV tet + CD4 + T cells in 7 healthy subjects was quantified as a percentage of tetramer + cells (C) or by frequency (D).Each symbol represents data from a distinct YFV-specific population.Experiments were repeated an average of 3.3 times.(E) The correlation between CD45RO -CCR7 + YFV tet + T cell frequency and the overall tet + frequency of the same population at least 7 months after vaccination.(F) The correlation between the CD45RO -CCR7 + YFV tet + T cell frequency and the fold change from the pre-vaccine baseline to the memory time point.CD45RO -CCR7 + tet + frequencies were determined using samples taken 7 to 34 months after vaccination.n = 33, populations with no post-vaccine naïve cells were excluded.RM one-way ANOVA (B) or Mixed-effect analysis (C and D) was performed and corrected with Tukey's multiple comparisons test.For (E) and (F), Pearson correlation was computed.

Figure 2 :
Figure 2: Post-vaccine CD4 + T cells are heterogeneous and include naïve-like subsets.(A) FACS plots show the expression of the indicated marker on a representative YFV-specific population.The tet + population is overlaid onto tet -bulk CD4 + T cells.(B) UMAP displays Phenograph-defined clusters.Data combine 1465 CD4 + cells labeled by 7 YFV tetramers from HD3. (C-D) The staining intensity of individual markers is shown on a heatmap for each cluster (C) or displayed on the UMAP.(E-F) The relative abundance of CD45RO - CCR7 + YFV-specific T cells by the indicated numbers of markers (E) or the type of markers (F).Frequency in F combines all cells positive for a particular marker within the CD45RO -CCR7 + subset.Marker combinations were determined using Boolean operators on manually defined gates.Each symbol represents a tetramer + population (n = 28).Experiments were repeated an average of 2.5 times.(G) PBMCs were stimulated for 4 -5 hours by PMA and ionomycin and assayed for cytokine production by intracellular cytokine staining.The plot shows representative TNF-a and IFN-g expression by TMN cells and memory T cells (non-CD45RO -CD28 + ) from the same tetramer-labeled population.(H) T cell responses by TNF-a and IFN-g production for the indicated phenotypic subset.Each population was identified with a pool of 5-7 tetramers of the same DR allele, using cells from 3 donors.For (E) and (F), RM one-way ANOVA was performed and corrected with Tukey's multiple comparison test.For (H), the Friedman test was performed and corrected using Dunn's multiple comparison test.

Figure 3 :
Figure 3: TMN-derived T cell clones respond to antigen stimulation.(A) Schematics of single-cell T cell cloning.Post-vaccine T cells from HD2 and HD3 were stained with 1501-YF45 tetramers, sorted based on TMN, TSCM, or TCM phenotypes, and expanded for 2 to 3 weeks in culture.(B) In vitro expanded T cell clones were restained with tetramers and cultured with vehicle or peptide-treated monocyte-derived dendritic cells.Representative plots show tetramer staining and cytokine production by intracellular cytokine staining.(C-D) T cell clones were stimulated with decreasing concentrations of YFV peptides.The response was measured by TNF-a production (C) and quantified by EC50 values after subtracting the background signal from vehicletreated control (D).(E) Peptide dose response of T cell clones by IFN-g, IL-2, and IL2 + TNF-a + IFN-g + production.(F) Representative histograms show CTV dilution in response to 10ug/ml of peptide stimulation.(G) Plot summarizes the frequency of CTVlow population after a 5-day culture for clones in each phenotypic group.All experiments were repeated at least twice with n= 5 in each group.For (C) and (E), RM two-way ANOVA was performed and corrected with Tukey's multiple comparison test.For (D) and (G), Kruskal-Wallis test and Dunn's multiple comparison test were used.

Figure 4 :
Figure 4: TMN cells are clonally related to memory T cells.(A) The plot summarizes the number of cells sequenced from the indicated specificities and donors collected 242 (HD2) or 210 (HD3) days after primary YFV vaccination.(B) Clone size and phenotypic distribution of YFV tet + populations in A. Phenotypic information were obtained by index sorting.TMN (CD45RO -CCR7 + CXCR3 -CD95 -CD11a -CD49d -), TSCM (CD45RO -CCR7 + and positive for at least one of CXCR3, CD95, CD11a, or CD49d), TCM (CD45RO + CCR7 + ), TEM (CD45RO + CCR7 -), TEMRA (CD45RO -CCR7 -).Cells with ambiguous phenotypes were excluded.(C) Distribution of phenotypes in B by clonotype frequency, ranked from largest to unique clonotypes.(D) The frequencies of YF45-specific T cells pre-vaccine and at the indicated days following YFV immunization.(E) Each circus plot represents TCRs from YF45 tet + cells obtained 210-242 days after vaccination, separated by the associated indexed phenotypes.Cells are ordered by frequency within each arc.Gray marks cells expressing unique TCRs, other colors represent expanded or shared sequences.Shared TCRb or TCRa and b, when a TCRa is available, is connected by a line across distinct phenotypic subsets.Blue lines highlight TCRs from TMN cells that are shared with cells expressing other phenotypes.(F) The percentage of TCRs in each memory subset that matched TMN-derived sequences.

Figure 5 :
Figure 5: TMN cells are clonally related to effector T cells and increased after vaccination.(A) The number of TCRs from YF45 tet + T cells from the indicated donors, before and 14 days after YFV vaccination.(B) TCRs from cells in A were compared to TMN-derived TCRs in the day 210-242 post-vaccine samples.Bargraphs show the percentages of cells with or without a match.(C) Each circus plot represents TCRs from TMN YF45 tet + cells and TCRs from the same specificities, before and 14 days after YFV vaccination, from the same donors.Cells are ordered by frequency within each arc.Gray marks cells expressing unique TCRs, other colors represent expanded or shared sequences.Connecting lines highlight shared TCRs between a TMN cell and cells in a previous time point.(D) The number and phenotypes of T cells in the day 14 sample that matched a TMN-derived clonotype from the memory time point.(E) Clonal dynamics of a TCR expressed by a TMN cell in the 14-day post-vaccine sample.Plot shows the number and phenotypes of T cells that expressed the same TCR sequence at the indicated time points.ND, not detected.(F-G) The frequencies of CD45RO - CCR7 + YFV-specific T cells before and after vaccination for the corresponding populations (F).Post-vaccine CD45RO -CCR7 + tet + cells were separated into TSCM or TMN subsets based on CXCR3, CD95, CD11a, or CD49d staining (G).(H) Plot compares the frequencies of CD45RO -CCR7 + tetramer + cells pre-vaccination with the frequencies of TMN cells in the corresponding population from the post-vaccine sample.(I) The post-vaccine TMN frequency of each tet + population was divided by its initial CD45RO -CCR7 + frequency before vaccination.The ratio is defined as a gain if above 1 and a loss if below 1. Pie chart shows the numbers of populations that had gained or lost naïve T cells after vaccination.(J) Plot shows the fold-change in tet + frequency, from the pre-vaccine baseline to the memory time point, for populations that had gained or lost naïve cells.For (F), (H), and (J), n = 27 tetramer + populations.Welch's t-test was performed.

Figure 6 :
Figure 6: TMN cells are stable and associated with durable T cell memory.(A) Representative plots show YFV-specific CD4 + T cells over the indicated time points from HD2. (B) Each tet + population of a given specificity was subdivided according to phenotypes.The change over time for each phenotypic subset was quantified by the estimated slope using a mixed effects exponential decay model (n = 19 tetramer + populations from 5 donors).(C) A mixed effects exponential decay model fitted to the dynamics of YFV-specific CD4 + T cells after a single YFV vaccination (n = 8 populations, combined from donors 4 and 5).The estimated decay (blue line) was used for calculating the half-life (t1/2).(D) Ranking of tet + populations by the averaged frequency of TMN cells within each population across all time points.(E) Plot summarizes the estimated slopes of individual tet + populations, divided into top and bottom halves by TMN frequency in D. (F) The correlation between slopes characterizing the change over time for the overall tetramer + populations and their corresponding averaged TMN frequencies.(G) Pie-charts show the distribution of memory subsets.Populations were divided into top and bottom groups by the first measured TMN frequency obtained within 1-2 years after YFV vaccination.(H) Phenotypic diversity of each tet + population was quantified using Shannon Diversity Index, categorized into top or bottom groups based on TMN frequency as in G.Each symbol represents one tetramer + population.Experiments were repeated an average of 2.3 times.Data are represented as mean ± SEM.For (B), RM one-way ANOVA was performed and corrected with Tukey's multiple comparisons test.Welch's t-test was performed for (E) and (H).For (F), Spearman correlation was performed.